Mg2+-dependent folding of a Diels-Alderase ribozyme probed by single-molecule FRET analysis

Abstract

Here, we report a single-molecule fluorescence resonance energy transfer (FRET) study of a Diels-Alderase (DAse) ribozyme, a 49-mer RNA with true catalytic properties. The DAse ribozyme was labeled with Cy3 and Cy5 as a FRET pair of dyes to observe intramolecular folding, which is a prerequisite for its recognition and turnover of two organic substrate molecules. FRET efficiency histograms and kinetic data were taken on a large number of surface-immobilized ribozyme molecules as a function of the Mg2+ concentration in the buffer solution. From these data, three separate states of the DAse ribozyme can be distinguished, the unfolded (U), intermediate (I) and folded (F) states. A thermodynamic model was developed to quantitatively analyze the dependence of these states on the Mg2+ concentration. The FRET data also provide information on structural properties. The I state shows a strongly cooperative compaction with increasing Mg2+ concentration that arises from association with several Mg2+ ions. This transition is followed by a second Mg2+-dependent cooperative transition to the F state. The observation of conformational heterogeneity and continuous fluctuations between the I and F states on the ∼100 ms timescale offers insight into the folding dynamics of this ribozyme.

Long-range tertiary interactions in single hammerhead ribozymes bias motional sampling toward catalytically active conformations

“…All our measured widths increase at high Mg 2+ concentrations (Fig. 3), consistent with both our previous work on minimal hammerheads and with data on a DielsAlderase ribozyme that likewise showed increased widths of intramolecular distance distributions with increasing Mg 2+ concentration (Kobitski et al 2007). Notably, the distribution of distances measured for the AFHH-WT hammerhead is significantly wider than those of the mutants (Fig.…”

Crystal structure of 3WJ core revealing divalent ion-promoted thermostability and assembly of the Phi29 hexameric motor pRNA

“…smFRET has been widely used to study the ion-induced conformational changes of functional RNAs (Zhuang et al 2000;Kobitski et al 2007;Qu et al 2008;Steiner et al 2008;Xiao et al 2008;Brenner et al 2010;McDowell et al 2010;Haller et al 2011). In this study, we investigated the effect of Mg 2+ on the folding and conformational change of pRNA 3WJ using smFRET.…”

RNA dynamics: it is about time

“…Clearly, slow global conformational dynamics significantly impact the overall catalytic rate constant [23••-25]. Single molecule FRET also allowed for the dissection of the metal-ion dependent multi-step folding pathways of an in vitro selected Diels-Alderase ribozyme [26] and a DNAzyme [27]. Sometimes FRET can demonstrate the lack of significant conformational dynamics of a ribozyme, for example of the glmS ribozyme upon cofactor binding [18].…”